Management of the refractory vitiligo patient: current therapeutic strategies and future options.

Vitiligo is an autoimmune disease that leads to disfiguring depigmented lesions of skin and mucosa. Although effective treatments are available for vitiligo, there are still some patients with poor responses to conventional treatment. Refractory vitiligo lesions are mostly located on exposed sites such as acral sites and lips, leading to significant life stress. Understanding the causes of refractory vitiligo and developing targeted treatments are essential to enhance vitiligo outcomes. In this review, we summarized recent treatment approaches and some potential methods for refractory vitiligo. Janus kinase inhibitors have shown efficacy in refractory vitiligo. A variety of surgical interventions and fractional carbon dioxide laser have been widely applied to combination therapies. Furthermore, melanocyte regeneration and activation therapies are potentially effective strategies. Patients with refractory vitiligo should be referred to psychological monitoring and interventions to reduce the potential pathogenic effects of chronic stress. Finally, methods for depigmentation and camouflage may be beneficial in achieving uniform skin color and improved quality of life. Our ultimate focus is to provide alternative options for refractory vitiligo and to bring inspiration to future research.

Viscoelastic parameterization of human skin cells characterize material behavior at multiple timescales.

Countless biophysical studies have sought distinct markers in the cellular mechanical response that could be linked to morphogenesis, homeostasis, and disease. Here, an iterative-fitting methodology visualizes the time-dependent viscoelastic behavior of human skin cells under physiologically relevant conditions. Past investigations often involved parameterizing elastic relationships and assuming purely Hertzian contact mechanics, which fails to properly account for the rich temporal information available. We demonstrate the performance superiority of the proposed iterative viscoelastic characterization method over standard open-search approaches. Our viscoelastic measurements revealed that 2D adherent metastatic melanoma cells exhibit reduced elasticity compared to their normal counterparts-melanocytes and fibroblasts, and are significantly less viscous than fibroblasts over timescales spanning three orders of magnitude. The measured loss angle indicates clear differential viscoelastic responses across multiple timescales between the measured cells. This method provides insight into the complex viscoelastic behavior of metastatic melanoma cells relevant to better understanding cancer metastasis and aggression.

Tfap2b specifies an embryonic melanocyte stem cell that retains adult multifate potential.

Melanocytes, the pigment-producing cells, are replenished from multiple stem cell niches in adult tissue. Although pigmentation traits are known risk factors for melanoma, we know little about melanocyte stem cell (McSC) populations other than hair follicle McSCs and lack key lineage markers with which to identify McSCs and study their function. Here we find that Tfap2b and a select set of target genes specify an McSC population at the dorsal root ganglia in zebrafish. Functionally, Tfap2b is required for only a few late-stage embryonic melanocytes, and is essential for McSC-dependent melanocyte regeneration. Fate mapping data reveal that tfap2b+ McSCs have multifate potential, and are the cells of origin for large patches of adult melanocytes, two other pigment cell types (iridophores and xanthophores), and nerve-associated cells. Hence, Tfap2b confers McSC identity in early development, distinguishing McSCs from other neural crest and pigment cell lineages, and retains multifate potential in the adult zebrafish.

SOX10: 20 years of phenotypic plurality and current understanding of its developmental function.

SOX10 belongs to a family of 20 SRY (sex-determining region Y)-related high mobility group box-containing (SOX) proteins, most of which contribute to cell type specification and differentiation of various lineages. The first clue that SOX10 is essential for development, especially in the neural crest, came with the discovery that heterozygous mutations occurring within and around SOX10 cause Waardenburg syndrome type 4. Since then, heterozygous mutations have been reported in Waardenburg syndrome type 2 (Waardenburg syndrome type without Hirschsprung disease), PCWH or PCW (peripheral demyelinating neuropathy, central dysmyelination, Waardenburg syndrome, with or without Hirschsprung disease), intestinal manifestations beyond Hirschsprung (ie, chronic intestinal pseudo-obstruction), Kallmann syndrome and cancer. All of these diseases are consistent with the regulatory role of SOX10 in various neural crest derivatives (melanocytes, the enteric nervous system, Schwann cells and olfactory ensheathing cells) and extraneural crest tissues (inner ear, oligodendrocytes). The recent evolution of medical practice in constitutional genetics has led to the identification of SOX10 variants in atypical contexts, such as isolated hearing loss or neurodevelopmental disorders, making them more difficult to classify in the absence of both a typical phenotype and specific expertise. Here, we report novel mutations and review those that have already been published and their functional consequences, along with current understanding of SOX10 function in the affected cell types identified through in vivo and in vitro models. We also discuss research options to increase our understanding of the origin of the observed phenotypic variability and improve the diagnosis and medical care of affected patients.

Phenotypic plasticity of melanocytes derived from human adult skin.

We previously described a novel in vitro culture technique for dedifferentiated human adult skin melanocytes. Melanocytes cultured in a defined, cholera toxin and PMA free medium became bipolar, unpigmented, and highly proliferative. Furthermore, TRP-1 and c-Kit expression disappeared and EGFR receptor and nestin expression were induced in the cells. Here, we further characterized the phenotype of these dedifferentiated cells and by comparing them to mature pigmented melanocytes we detected crucial steps in their phenotype change. Our data suggest that normal adult melanocytes easily dedifferentiate into pluripotent stem cells given the right environment. This dedifferentiation process described here for normal melanocyte is very similar to what has been described for melanoma cells, indicating that phenotype switching driven by environmental factors is a general characteristic of melanocytes that can occur independent of malignant transformation.

Activation of cGMP-Dependent Protein Kinase Restricts Melanoma Growth and Invasion by Interfering with the EGF/EGFR Pathway.

Drug resistance mechanisms still characterize metastatic melanoma, despite the new treatments that have been recently developed. Targeting of the cGMP/protein kinase G pathway is emerging as a therapeutic approach in cancer research. In this study, we evaluated the anticancer effects of two polymeric-linked dimeric cGMP analogs able to bind and activate protein kinase G, called protein kinase G activators (PAs) 4 and 5. PA5 was identified as the most effective compound on melanoma cell lines as well as on patient-derived metastatic melanoma cells cultured as three-dimensional spheroids and in a zebrafish melanoma model. PA5 was able to significantly reduce cell viability, size, and invasion of melanoma spheroids. Importantly, PA5 showed a tumor-specific outcome because no toxic effect was observed in healthy melanocytes exposed to the cGMP analog. We defined that by triggering protein kinase G, PA5 interfered with the EGF pathway as shown by lower EGFR phosphorylation and reduction of activated, phosphorylated forms of protein kinase B and extracellular signal‒regulated kinase 1/2 in melanoma cells. Finally, PA5 significantly reduced the metastatic process in zebrafish. These studies open future perspectives for the cGMP analog PA5 as a potential therapeutic strategy for melanoma.

BRAFV600E induces reversible mitotic arrest in human melanocytes via microrna-mediated suppression of AURKB.

Benign melanocytic nevi frequently emerge when an acquired BRAFV600E mutation triggers unchecked proliferation and subsequent arrest in melanocytes. Recent observations have challenged the role of oncogene-induced senescence in melanocytic nevus formation, necessitating investigations into alternative mechanisms for the establishment and maintenance of proliferation arrest in nevi. We compared the transcriptomes of melanocytes from healthy human skin, nevi, and melanomas arising from nevi and identified a set of microRNAs as highly expressed nevus-enriched transcripts. Two of these microRNAs-MIR211-5p and MIR328-3p-induced mitotic failure, genome duplication, and proliferation arrest in human melanocytes through convergent targeting of AURKB. We demonstrate that BRAFV600E induces a similar proliferation arrest in primary human melanocytes that is both reversible and conditional. Specifically, BRAFV600E expression stimulates either arrest or proliferation depending on the differentiation state of the melanocyte. We report genome duplication in human melanocytic nevi, reciprocal expression of AURKB and microRNAs in nevi and melanomas, and rescue of arrested human nevus cells with AURKB expression. Taken together, our data describe an alternative molecular mechanism for melanocytic nevus formation that is congruent with both experimental and clinical observations.

Lots of people have small dark patches on their skin known as moles. Most moles form when individual cells known as melanocytes in the skin acquire a specific genetic mutation in a gene called BRAF. This mutation causes the cells to divide rapidly to form the mole. After a while, most moles stop growing and remain harmless for the rest of a person's life. Melanoma is a type of skin cancer that develops from damaged melanocytes. The same mutation in BRAF that is found in moles is also present in half of all cases of melanoma. Unlike in moles, the melanoma-causing mutation makes the melanocytes divide rapidly to form a tumor that keeps on growing indefinitely. It remains unclear why the same genetic mutation in the BRAF gene has such different consequences in moles and melanomas. To address this question, McNeal et al. used genetic approaches to study melanocytes from moles and melanomas. The experiments identified some molecules known as microRNAs that are present at higher levels in moles than in melanomas. Increasing the levels of two of these microRNAs in melanocytes from human skin stopped the cells from growing and dividing by inhibiting a gene called AURKB. This suggested that these microRNAs are responsible for halting the growth of moles. Introducing the mutated form of BRAF into melanocytes also stopped cells from growing and dividing by inhibiting AURKB. However, changing the environment surrounding the cells reversed this effect and allowed the melanocytes to resume dividing. In this way the mutated form of BRAF acts like a switch that allows melanocytes in skin cancers to start growing again under certain conditions. Further experiments found that a drug called barasertib is able to inhibit the growth of melanoma cells with the mutant form of BRAF. Future work will investigate whether it is possible to use this drug and other tools to stop skin cancer tumors from growing, and possibly even prevent skin tumors from forming in the first place.

Nerve-associated Schwann cell precursors contribute extracutaneous melanocytes to the heart, inner ear, supraorbital locations and brain meninges.

Melanocytes are pigmented cells residing mostly in the skin and hair follicles of vertebrates, where they contribute to colouration and protection against UV-B radiation. However, the spectrum of their functions reaches far beyond that. For instance, these pigment-producing cells are found inside the inner ear, where they contribute to the hearing function, and in the heart, where they are involved in the electrical conductivity and support the stiffness of cardiac valves. The embryonic origin of such extracutaneous melanocytes is not clear. We took advantage of lineage-tracing experiments combined with 3D visualizations and gene knockout strategies to address this long-standing question. We revealed that Schwann cell precursors are recruited from the local innervation during embryonic development and give rise to extracutaneous melanocytes in the heart, brain meninges, inner ear, and other locations. In embryos with a knockout of the EdnrB receptor, a condition imitating Waardenburg syndrome, we observed only nerve-associated melanoblasts, which failed to detach from the nerves and to enter the inner ear. Finally, we looked into the evolutionary aspects of extracutaneous melanocytes and found that pigment cells are associated mainly with nerves and blood vessels in amphibians and fish. This new knowledge of the nerve-dependent origin of extracutaneous pigment cells might be directly relevant to the formation of extracutaneous melanoma in humans.

Melanocytes determine angiogenesis gene expression across human tissues.

Several angiogenesis-dependent diseases, including age-related macular degeneration and infantile hemangioma, display differential prevalence among Black, as compared to White individuals. Although socioeconomic status and genetic architecture have been suggested as explaining these differences, we have recently shown that pigment production per se might be involved. For example, we have shown that the extracellular protein fibromodulin is a pro-angiogenic factor highly secreted by melanocytes in White but not Black individuals. Still, additional pigment-dependent angiogenic factors and their molecular mechanisms remain to be identified. Understanding the contribution of pigmentation to angiogenesis in health and disease is essential for precision medicine of angiogenesis-dependent diseases with racial disparity. Toward that goal, we compared the transcriptomes of Black and White individuals in three tissues with angiogenic activity, namely artery, whole blood, and skin. We identified several differentially expressed angiogenesis pathways, including artery morphogenesis, regulation of endothelial cell chemotaxis, and cellular response to vascular endothelial growth factor stimulus. We then demonstrated that the expression of key genes in these pathways is directly modulated by the degree of pigmentation. We further identified the precise pigment production pathway controlling the expression of these genes, namely melanocortin 1 receptor (MC1R) signaling. These results demonstrate pigment-mediated regulation of angiogenesis-related pathways and their driver genes across human tissues.

Long non-coding RNA Rian promotes the expression of tight junction proteins in endothelial cells by regulating perivascular-resident macrophage-like melanocytes and PEDF secretion.

Perivascular-resident macrophage-like melanocytes (PVM/Ms) can upregulate the expression of tight junction-related proteins in endothelial cells (ECs) by secreting pigment epithelial-derived factor (PEDF), and thereby regulate the permeability of the intrastrial fluid-blood barrier critical for maintaining inner ear homeostasis. This study aimed to investigate the effects of long non-coding RNA (lncRNA) Rian on cell growth of PVM/Ms and PVM/Ms regulation of intrastrial fluid-blood barrier integrity mediated by PEDF. Rian was downregulated in the aged cochlea from 12-month-old C57BL/6 mice. Rian overexpression inhibited cell apoptosis and promoted cell viability of hypoxia-injured PVM/Ms as well as increased the concentration and expression of PEDF secreted by PVM/Ms. In contrast, Rian silencing exerted the opposite effects. Furthermore, in a cell co-culture model of ECs and PVM/Ms, Rian overexpression in PVM/Ms increased the expression of the junction-associated proteins in co-cultured ECs, and this effect was abrogated by blockade of PEDF by anti-PEDF in PVM/Ms. Further mechanistical investigation revealed that Rian promoted STAT3 nuclear translocation and activation by binding to FUS, and thereby promoted the secretion of PEDF. Collectively, Rian attenuates PVM/Ms injury and strengthens the ability of PVM/Ms to maintain the integrity of the endothelial barrier by promoting PEDF expression.

Biology of Melanoma.

Melanoma skin cancer is derived from skin melanocytes and has a high risk of metastatic spread. The era of molecular genetics and next-generation sequencing has uncovered the role of oncogenic BRAFV600E mutations in many melanomas, validated the role of ultraviolet-induced DNA mutations in melanoma formation, and uncovered many of the molecular events that occur during melanoma development. Targeted therapies and immunotherapy have dramatically improved outcomes and provided an increased rate of cure for metastatic melanoma. This article reviews the formation of melanoma, the molecular events involved in melanoma growth and metastasis, and the biology underlying resistance to melanoma therapies.

Cyclic adenosine monophosphate (cAMP) signaling in melanocyte pigmentation and melanomagenesis.

The second messenger cyclic adenosine monophosphate (cAMP) regulates numerous functions in both benign melanocytes and melanoma cells. cAMP is generated from two distinct sources, transmembrane and soluble adenylyl cyclases (tmAC and sAC, respectively), and is degraded by a family of proteins called phosphodiesterases (PDEs). cAMP signaling can be regulated in many different ways and can lead to varied effects in melanocytes. It was recently revealed that distinct cAMP signaling pathways regulate pigmentation by either altering pigment gene expression or the pH of melanosomes. In the context of melanoma, many studies report seemingly contradictory roles for cAMP in tumorigenesis. For example, cAMP signaling has been implicated in both cancer promotion and suppression, as well as both therapy resistance and sensitization. This conundrum in the field may be explained by the fact that cAMP signals in discrete microdomains and each microdomain can mediate differential cellular functions. Here, we review the role of cAMP signaling microdomains in benign melanocyte biology, focusing on pigmentation, and in melanomagenesis.

Skin pigmentation and its control: From ultraviolet radiation to stem cells.

In the light of substantial discoveries in epithelial and hair pigmentation pathophysiology, this review summarizes the current understanding of skin pigmentation mechanisms. Melanocytes are pigment-producing cells, and their key regulating transcription factor is the melanocyte-specific microphthalmia-associated transcription factor (m-MITF). Ultraviolet (UV) radiation is a unique modulator of skin pigmentation influencing tanning pathways. The delayed tanning pathway occurs as UVB produces keratinocyte DNA damage, causing p53-mediated expression of the pro-opiomelanocortin (POMC) gene that is processed to release α-melanocyte-stimulating hormone (α-MSH). α-MSH stimulates the melanocortin 1 receptor (MC1R) on melanocytes, leading to m-MITF expression and melanogenesis. POMC cleavage also releases ß-endorphin, which creates a neuroendocrine pathway that promotes UV-seeking behaviours. Mutations along the tanning pathway can affect pigmentation and increase the risk of skin malignancies. MC1R variants have received considerable attention, yet the allele is highly polymorphic with varied phenotypes. Vitiligo presents with depigmented skin lesions due to autoimmune destruction of melanocytes. UVB phototherapy stimulates melanocyte stem cells in the hair bulge to undergo differentiation and upwards migration resulting in perifollicular repigmentation of vitiliginous lesions, which is under sophisticated signalling control. Melanocyte stem cells, normally quiescent, undergo cyclic activation/differentiation and downward migration with the hair cycle, providing pigment to hair follicles. Physiological hair greying results from progressive loss of melanocyte stem cells and can be accelerated by acute stress-induced, sympathetic driven hyperproliferation of the melanocyte stem cells. Ultimately, by reviewing the pathways governing epithelial and follicular pigmentation, numerous areas of future research and potential points of intervention are highlighted.

Borage Oil Treated with Immobilized Lipase Inhibits Melanogenesis.

In the present study, we demonstrated that borage (Borago officinalis L.) seed oil subjected to immobilized lipase pretreatment are enriched with linoleic acid (LNA, 18:2n-6), γ-linolenic acid (GLA, 18:3n-6), and oleic acid (OLA, 18:1n-9). We further showed that lipase-treated borage oil (LT-BOL) regulates the activity and degradation of tyrosinase, an important enzyme implicated in the synthesis of melanin in murine melanocytes, B16F10. LT-BOL and its free fatty acid components reduced the levels of melanin and tyrosinase in melanocytes with GLA exerting similar or stronger effects compared with LNA and OLA. The brightening efficacy of LT-BOL on melanin metabolism in humans was tested by an 8-week, double-blind, randomized clinical trial, which enrolled 21 Korean female adults (mean age 48.57 ± 3.28). Visual evaluation showed that cream containing 1% LT-BOL significantly decreased (p < 0.05) melasma on the treated skin area after 6 and 8 weeks. The analysis of the skin brightness using Chromameter CR-400 confirmed that the brightness of the treated area was significantly increased (p < 0.01) after 4, 6, and 8 weeks. Together, our results suggest that LT-BOL may be suitable as a natural skin whitening cosmeceutical product.

The Role of Autophagy in Skin Fibroblasts, Keratinocytes, Melanocytes, and Epidermal Stem Cells.

Human skin acts as a barrier to protect our bodies from UV rays and external pathogens and to prevent water loss. Phenotypes of aging, or natural aging due to chronic damage, include wrinkles and the reduction of skin thickness that occur because of a loss of skin cell function. The dysregulation of autophagy, a lysosome-related degradation pathway, can lead to cell senescence, cancer, and various human diseases due to abnormal cellular homeostasis. Here, we discuss the roles and molecular mechanisms of autophagy involved in the anti-aging effects of autophagy and the relationship between autophagy and aging in skin cells.

A new model to investigate UVB-induced cellular senescence and pigmentation in melanocytes.

Ultraviolet (UV) light is known to potentially damage human skin and accelerate the skin aging process. Upon UVB exposure, melanocytes execute skin protection by increasing melanin production. Senescent cells, including senescent melanocytes, are known to accumulate in aged skin and contribute to the age-associated decline of tissue function. However, melanocyte senescence is still insufficiently explored. Here we describe a new model to investigate mechanisms of UVB-induced senescence in melanocytes and its role in photoaging. Exposure to mild and repeated doses of UVB directly influenced melanocyte proliferation, morphology and ploidy. We confirmed UVB-induced senescence with increased senescence-associated ß-galactosidase positivity and changed expression of several senescence markers, including p21, p53 and Lamin B1. UVB irradiation impaired proteasome and increased autophagic activity in melanocytes, while expanding intracellular melanin content. In addition, using a co-culture system, we could confirm that senescence-associated secretory phenotype components secreted by senescent fibroblasts modulated melanogenesis. In conclusion, our new model serves as an important tool to explore UVB-induced melanocyte senescence and its involvement in photoaging and skin pigmentation.

H2 O2 -induced oxidative stress disrupts mitochondrial functions and impairs migratory potential of human epidermal melanocytes.

Reactive oxygen species (ROS) have already been demonstrated to impede the migratory ability in non-melanocytic cell lines by depleting mitochondrial ATP production. Therefore, understanding the mitochondrial metabolic response to migration in the presence of ROS should be a key to understanding repigmentation in vitiligo. This study aimed to investigate the energy mechanism associated with the ROS-mediated attenuation of melanocyte migration. After melanocytes were pretreated with H2 O2 , their ATP production, migratory ability, ultrastructural changes and Mitochondrial Permeability Potential were analysed. The results showed that, in parallel with the decreased ATP production, the migratory ability of melanocytes was significantly inhibited by oxidative stress. Supplementation with exogenous ATP reversed the suppressed ATP-dependent migration of melanocytes. Melanocytes were then stressed with H2 O2 and Agilent Whole Human Genome microarray analysis identified 763 up-regulated mRNAs and 1117 down-regulated mRNAs. Among them, 11 of the encoded proteins were involved in mitochondrial ATP production and their expression levels were verified. The decreased expression of NADH dehydrogenase 2(ND2) , cytochrome c oxidase 1(COX1) and cytochrome c oxidase 3(COX3) was shown to be involved in the depletion of mitochondrial ATP production, which was coupled with the impaired migratory potential. These results indicate that the migration of melanocytes relies heavily on an inexhaustible supply of ATP from mitochondria.

Aberrant ETS-1 signalling impedes the expression of cell adhesion molecules and matrix metalloproteinases in non-segmental vitiligo.

Cell adhesion is a complex process that involves multiple molecules on the cell surface (ie cell adhesion molecules [CAMs]), surrounding cells and extracellular matrix (ECM). Repigmentation in vitiligo is dependent on the ECM remodelling and cellular migration, primarily attributed to the transcriptional activation of matrix metalloproteinases (MMPs). In this study, we aimed to demonstrate the role of ETS-1 signalling in the regulation of MMPs and CAMs. Therefore, we studied the expression of ETS-1, MMPs (MMP-2, MMP-9) and CAMs including E-cadherin, ITGA-1 and ICAM-1 in vitiligo (both active and stable) ex vivo. Further, we compared melanocyte morphology and their adhesion towards collagen IV and laminin between control and vitiligo groups in vitro. Also, we silenced ETS-1 in melanocytes cultured from control skin and observed post-silencing effect on above-mentioned MMPs and CAMs. We perceived absent ETS-1 and significantly reduced CAMs and MMPs in vitiligo compared with normal skin. Scanning electron microscopy (SEM) revealed a translucent material surrounding individual melanocytes in stable vitiligo and controls, whereas active vitiligo melanocytes demonstrated loss of this extracellular substance. Adhesion assays revealed significantly decreased binding of cultured melanocytes to collagen IV and laminin V plates in both stable and active vitiligo. Importantly, ETS-1 silencing resulted in significantly reduced expression of CAMs and MMPs. In conclusion, absent ETS-1 expression in both stable and active non-segmental vitiligo seems to impede the expression of CAMs, apart from MMPs, probably leading to progressive depigmentation in active disease and absence of spontaneous repigmentation in stable disease.

Tranexamic Acid Inhibits Angiogenesis and Melanogenesis in Vitro by Targeting VEGF Receptors.

Melasma is a common but complex skin condition concerning cosmetic problems. Tranexamic acid (TA) has been proved to be effective in treatment of melasma with still unclear mechanisms. Here, we show that VEGF165 enhanced the expression of VEGF receptors (VEGFRs, including VEGFR-1, VEGFR-2 and NRP-1) in human umbilical vein endothelial cells (HUVECs), which was attenuated by TA. VEGF165 also promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2 in HUVECs, which was again abolished by TA. TA further showed similar effects to neutralization of VEGFR-1 and VEGFR-2 in inhibiting cell proliferation, migration, invasion and tube formation of HUVECs induced by VEGF165, suggesting that TA could inhibit angiogenesis by targeting VEGFRs in HUVECs. In addition, VEGF165 enhanced the expression of VEGFRs and promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2 in normal human melanocytes, which were also attenuated by TA. Furthermore, TA showed similar effects to neutralization of VEGFR-1 and VEGFR-2 in inhibiting tyrosinase activity, melanin production and even melanogenic proteins induced by VEGF165, suggesting that TA could reduce melanogenesis via inhibiting activation of VEGFRs and subsequent expression of melanogenic proteins in melanocytes. Taken together, we demonstrate that TA can inhibit angiogenesis and melanogenesis in vitro at least in part by targeting VEGFRs, which may offer a new understanding of the pathogenesis of melasma as well as the molecular mechanism for TA in treatment of the disease.